Isolated extract of walnuts, method for its obtention and its use

ABSTRACT

The invention refers to an isolated extract of walnuts of high efficiency and stability over time useful for the manufacture of a medicament for the treatment of viral, fungal and bacterial diseases, which is characterized in that it is obtained from a process comprising: i) collecting unripe walnut fruits as raw material; ii) preparing the raw material for the extraction, iii) freezing the raw material prepared in the previous step; iv) drying; v) extracting in a time lower than 10 minutes; vi) filtration; and vii) final packaging. The invention also refers to its use for manufacturing a medicament for the treatment of bacterial, fungal and viral diseases and to a composition comprising it.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/302,725, filed on Nov. 26, 2008, which is a U.S. nationalapplication under 35 U.S.C. §371 of PCT Application Serial No.PCT/EP2007/055176 filed on May 29, 2007, which claims priority toSpanish Patent Application No. P-200601542, filed Jun. 2, 2006, thedisclosures of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention refers to an isolated extract of walnuts with ahigh efficiency and stability over time useful for the manufacture of amedicament for the treatment of viral, fungal and bacterial diseases.

The present invention also refers to a process for obtaining saidisolated extract of walnuts.

BACKGROUND OF THE INVENTION

The walnut extracts and cataplasms have been used during many years inpopular dietetic medicaments.

The walnut tree contains at the bark, leafs or fruits gallic andcatequic tannins, juglone, juglandine, carotene, inositol, pyrogallol,vitamin C and other substances.

It has been demonstrated that the walnut tree bark (Manchurian) containsmainly 5-hydroxi-1,4-naphtoquinones (juglone),1,4,5-trihydroxinaphtalenes (hydrojuglone), glycosides of hydrojuglone,glycosides of hydroxi-α-tetralone and gallic derivatives of theseglycosides. It is also known that the walnut hull is very rich invitamin C and that betacarotene, B1, B2 and B6 have been found in theleaves.

The juglone is considered as a natural product having anti-microbial,anti-neoplastic and anti-parasitic action and has anti-fungal andantiseptic properties. Therefore, it can be found in different marketproducts, including colorant compositions for hair and oil of satinwalnuts. The juglone, prepared from walnut hull, is an active ingredientactive in dietetic complements.

There are evidences that suggest that the juglone is a strongchemotherapeutic or chemopreventive agent. The therapeutic developmentProgram, National Cancer Institute (NCI) evaluated the juglone in itssearch for the HIV-1.

U.S. Pat. No. 6,296,838, from October 2001, refers to an anti-fungalcomposition, of topical application, for the treatment and prevention ofthe human nails infected with fungi. In particular, U.S. Pat. No.6,296,838 describes that the extract with synergic effects contains amixture of 10-15% of juglone extract from walnut hull with 20-30% ofmilled roots of Nardostachys jatamansi o Vetiveria zizanioides oCatharanthus roseus, because an extract of only juglone showed verylittle efficiency even in the long curing treatment and fungi-toxiceffect.

To obtain said extract the walnut hulls were separated, washed withwater and air dried. Then, they were milled and an extraction of juglonewith solvents selected from acetone, alcohol and butanol was carriedout. The solvent was removed from the extract and it was cooled toremove the waxes so that the juglone was partially fractionated.

The isolated extracts of walnuts described in the state of the art arebased in the extraction of determined active ingredients with ananti-fungal, anti-microbial or anti-viral purpose, from a part of thetree whose active ingredient is found in great quantities.

There are anti-fungal compositions containing extract of juglone fromwalnut hull or compositions with a high concentration of vitamin Cobtained from the tissues, walnut tree leaves, with a great content ofvitamin C, among some of the most known.

The compositions comprising isolated extracts of walnuts present thedrawback that it must be administered immediately after its preparation,said compositions having a short life because of the decrease of itseffectiveness over time.

Therefore, because of the low stability of the isolated extracts ofwalnuts, the compositions containing them must be produced andadministered within few days in order to provide a suitableeffectiveness in the treatment.

Thus, said compositions based in isolated extracts of walnut have alimited application over time, because of the low stability of thejuglone in the extract, and they are exclusively of anti-fungal,anti-microbial or anti-viral type.

Therefore, there has not provided yet any isolated extract from onlywalnuts which are stable over time and independent of the moment ofpreparation. Additionally, there has not been provided yet anycomposition containing said extract showing a simultaneous anti-fungal,anti-microbial and anti-viral application, whose properties are keptstable over time.

DESCRIPTION OF THE PRESENT INVENTION

A first aspect of the present invention is, therefore, to provide awalnut-based isolated extract stable over time and independent from themoment it was prepared.

A second aspect of the present invention is to provide a walnut-basedisolated extract which is simultaneously effective in the treatment offungi, bacteria and virus.

A third aspect of the invention is to provide a process permitting toobtain an isolated extract of walnuts according to the first and secondaspects of the invention.

A fourth aspect of the present invention is the use of an extractaccording to the first aspect of the present invention for themanufacture of a medicament for the treatment of viral, fungal andbacterial diseases.

A fifth aspect of the invention is to provide a composition comprisingan extract according to the first aspect of the invention to be used inthe manufacture of a medicament for the treatment of viral, fungal andbacterial diseases.

DEFINITIONS

In the present invention by “unripe fruits of walnuts” is meant thatsaid walnuts are in a Lactic-Waxy ripeness state. In general, saidripeness state takes places between 10th of June and 15th of September,more approximately from 10th of July and 10th of August, even thoughsaid dates can vary a little according to the weather of the place wherethe fruit is ripening.

FIGURES

FIG. 1 shows a flow chart of the process according to the presentinvention where the obtention of a walnut extract with an extractiontime lower than 10 min. is carried out.

FIG. 2 refers to a graphical representation of the content of juglone in(%) in a walnut extract in respect with the time from its preparation,as a function of the time used during the extraction step. From saidFIG. 2 it can be seen that the content of juglone was kept stable overtime only when the extraction time is lower than 10 min. (—▪—).

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have found that the extractiontime used during a walnut extract is obtained is a decisive condition inthe final properties of the obtained extract.

Advantageously, the isolated extract of walnuts according to the firstaspect of the invention shows properties for a simultaneous anti-fungal,anti-microbial and anti-viral application which is stable over time.

According to the three first aspects of the invention, there is providedan isolated extract of walnuts which characterized in that it isobtained from a process comprising:

-   -   i) Collecting unripe walnut fruits as raw material;    -   ii) Preparing the raw material for the extraction;    -   iii) Freezing the raw material prepared in the previous step;    -   iv) Drying;    -   v) Extracting in a time lower than 10 minutes;    -   vi) Filtration; and    -   vii) Final packaging.

In particular, the preparation of the raw material for the extraction(ii) comprises preferably a mechanical cleaning, a washing with runningwater, a washing with distilled water and a drying at the air.

Preferably, the freezing step (iii) comprises keeping the walnut fruitsin a frozen chamber at a temperature comprised between −18 and −20° C.during 70-170 hours, even more preferably during about 120±5 hours.

In particular, the drying step (iv) comprises, first, peeling the shellsof the frozen walnut in thin layers of 2 to 3 mm and, then, place themin trays to leave them dry, preferably under the sun and then in adrying chamber at a temperature of 38° C.±2° C. until a humidity lowerthan 10% is obtained.

The extraction step (v) comprises breaking the obtained dried mass, inthe drying step, into little pieces, and adding ethyl alcohol (96%) in a1/2 to 1/5 weight/volume ratio, preferably 1/3, and carrying out anextraction during a time lower than 10 minutes, preferably lower than 5minutes, even more preferably lower than 2 minutes.

The filtration step of the extract is carried out preferably in aprimary depuration filter and, then, in a bactericide filter of thinpore at a pressure of 0.25±0.005 MPa.

There are also included within the scope of the present invention thosemodifications that a person skilled in the art can carry out in thesteps i) to iv), vi) and vii), providing that they do not depart fromthe inventive concept object of the present invention. As a non-limitingdiagram of the invention of the process defined above see FIG. 1.

The inventors of the present invention have found that the extractiontime is critical in the final properties of the isolated extract ofwalnuts. It is believed that when the extraction time is increased,other components are removed from the walnut that possibly interfere inthe stability and efficiency over time of the obtained extract.

Surprisingly, the inventors of the present invention have found that theisolated extract of walnuts obtained according to the first aspect ofthe invention retains its properties for a simultaneous anti-fungal,anti-microbial and anti-viral application, apart from being stable overtime.

Therefore, the invention also provides a process for obtaining anisolated extract of walnuts as defined above according to the firstaspect of the invention.

The inventors of the present invention evaluated the stability of thejuglone as a function of the time used during the extraction step v).

In Table 1 below it can be seen that the longer extraction time is usedin step v), the lower is the stability over time of the activeingredient juglone.

TABLE 1 Months Assay Extraction 15 No. Time (h) 0 days 1 3 6 12 18 24 1<10 min 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 2  1 0.4 0.08 0.03 0 0 00 0 3  3 0.5 0.06 0.02 0 0 0 0 0 4  12 0.5 0.05 0.01 0 0 0 0 0 5  24 0.50.09 0.03 0 0 0 0 0

From the results shown in Table 1, it can be seen that the content ofjuglone in the final extract decreased when the extraction time of stepv) is increased, and also decreased the stability of the juglone extractover time after its preparation.

Surprisingly, the juglone extract obtained using an extraction timelower than 10 min was kept stable after its preparation during a time upto 24 months and, therefore, its efficiency did not depend on the timefrom its preparation, but the time used during the extraction time, seeassay No. 1 and FIG. 2.

Advantageously, with the isolated extract of juglone according to thefirst aspect of the present invention an extract is obtained with astability of more than two years, stability substantially longercompared to the walnut extracts disclosed in the state of the art ofapproximately some days.

The isolated extract of walnuts according to the present invention hasapplication in the treatment of herpes, chickenpox, fungi on the skin,nails, etc., in general in the treatment of skin diseases.

The administration of the extract according to the present invention canbe carried out by topical, intraperitoneal or oral application such as,e.g. liquid form, creams or tablets, even though other ways ofapplication of the extract according to the present invention are alsoincluded in the scope thereof.

The inventors of the present invention also studied the efficiency ofthe isolated extract according to the first aspect of the invention inbacteria, fungi and virus of the assays No. 1 to 5 (Table 1). See Table2 below.

TABLE 2 Extraction time <10 min 1 h 3 h 12 h 24 h Months Type (No. 1)(No. 2) (No. 3) (No. 4) (No. 5) 0 Staphylococus (+) (+) (+) (+) (+) ATCC6538 Pseudomonas (+) (+) (+) (+) (+) aeruginosa ATCC 9027 Aspergillus(+) (+) (+) (+) (+) niger ATCC 16404 Candida (+) (+) (+) (+) (+)albicans ATCC 10231 Herpes simplex (+*) (+*) (+*) (+*) (+*) virus type 1HSV-1 6 Staphylococus (+) (−) (−) (−) (−) ATCC 6538 Pseudomonas (+) (−)(−) (−) (−) aeruginosa ATCC 9027 Aspergillus (+) (−) (−) (−) (−) nigerATCC 16404 Candida (+) (+) (+) (+) (+) albicans ATCC 10231 Herpessimplex (+*) (−*) (−*) (−*) (−*) virus type 1 HSV-1 12 Staphylococus (+)(−) (−) (−) (−) ATCC 6538 Pseudomonas (+) (−) (−) (−) (−) aeruginosaATCC 9027 Aspergillus (+) (−) (−) (−) (−) niger ATCC 16404 Candida (+)(+) (+) (+) (+) albicans ATCC 10231 Herpes simplex (+*) (−*) (−*) (−*)(−*) virus type 1 HSV-1 24 Staphylococus (+) (−) (−) (−) (−) ATCC 6538Pseudomonas (+) (−) (−) (−) (−) aeruginosa ATCC 9027 Aspergillus (+) (−)(−) (−) (−) niger ATCC 16404 Candida (+) (+) (+) (+) (+) albicans ATCC10231 Herpes simplex (+*) (−*) (−*) (−*) (−*) virus type 1 HSV-1 TableFoot: (+) Depresses Growth; (−) Not Depresses Growth; (+*) DepressesReproduction of HSV-1 virus in DR cell cultures (bone sarcoma) and ofhuman musculocutaneos fibroblasts; (−*) Not Depresses Reproduction ofHSV-1 virus in DR cell cultures (bone sarcoma) and of humanmusculocutaneos fibroblasts;

From the data shown in the previous table it can be seen that when theextraction time was increased, the extract lost efficiency over timefrom the moment of its production.

The shown results prove that the isolated extract of walnuts accordingto the first aspect of the invention retains its efficiency over time inbacteria, fungi and virus up to 24 months from its preparation and thatsaid efficiency depends on the time used during the extraction step(assay No. 1).

Therefore, according to the fourth aspect of the present invention thereis also provided an isolated extract of walnuts which is used for themanufacture of a medicament for the treatment of viral, fungal andbacterial diseases.

The invention also refers to a composition comprising an extractaccording to the first aspect of the invention and to its use for themanufacture of a medicament for the treatment of viral, fungal andbacterial diseases.

Hereinafter some cytoxicity studies proving that the extract accordingto the first aspect of the present invention is suitable to be used inhumans are included.

General Toxicity Studies

The toxicological evaluation of the isolated extract of walnutsaccording to the first aspect (hereinafter, the extract) was carried outin rats and mice to which different concentrations of the extract wereintragastrically administered. The intragastrical administration wasproposed in order to obtain a systemic effect on the herpes virus, bothin cases of acute infection and in virus carriers. The extract can beconsidered, hence, a natural antiviral chemotherapeutical remedy.However, the manifestation of its toxic properties was expected becauseit was administered by systemic absorption, which was also confirmed ina sub-acute study with rats and mice. It must be pointed out, however,that the frequency of fatal ends, even when maximum doses areadministered intragastrically, is so low that it is impossible toestablish average lethal doses (LD₅₀, etc.). The extract has neither theproperty of material accumulation nor a mutagenic effect on bone marrowcells.

About the general toxic properties, it is necessary to point out theeffect on CNS and the inhibitory action on the growing of the body massin baby animals. The toxicity related to the parenchymatous organsconsists of, in particular, the deterioration of the parenchymatouscells of the liver and to the alteration of the excretory function ofkidneys. The reduction of the spleen and adrenals mass seems to reflectthe stress state as a result of the long-term manipulations for 30 days(see table 1.1, ethanol 5%).

Likewise, the extract doses which cause minimum deviations of the normalindexes in rats have been established, and this has been takenconventionally as the maximum tolerable dose administering 0.5 ml of0.5% solution for 30 days. In humans, this is equivalent to 250-500 mlof solution at said concentration. Actually, as it is well-known, inclinical conditions 1/100- 1/10 of the minimum toxic dose or “inactive”dose are assayed, which represents 2.5-5.0 ml of 0.5% solution per day.

In conclusion, the results of the toxicological evaluation of thesolutions of Ext at different concentrations applied cutaneously severaltimes show that the preparation at a dose of 0.2 ml of 5% solution doesnot cause evident intoxication effects. However, histologically, certaingrade of intensification of the keratinization process in the epidermisis shown without any increase of the layers of the epidermic cells andwithout inflammatory effects on the skin itself (the dermis).

The 50% extract solution produces evident hyperkeratosis, dryness andhardening of the cutaneous folds. Histologically, together with theintensification of the keratinisation process, an increase of the celllayers in the epidermis is also produced, even though in this case noinflammatory effects have been observed. With this dose no systemictoxicity (by absorption) signs have been observed.

In two weeks long experiment with mice, the animals received volumes ofabout 4 times greater than the solutions at the same concentrations (5and 50% solutions). The visual cutaneous reaction in a shedding andthickening manner of the cutaneous folds, histologically confirmed,without inflammatory effects, is already shown when using 5% Ext. Agranulocytopoietic reaction is also seen, shown in the transformation ofthe “lymphocytary” type, of hemopoiesis, characteristic of the rodents,to “granulocytary” type. The preparation at a concentration of 5% didnot produce any destructive or inflammatory change in the mucousmembrane of the distal segment of the rectum. At this dose no symptom ofa general systemic toxic effect was observed. The application on theskin of extract at a extremely high concentration (50%) is accompaniedby systemic toxicity effects. The characteristic properties of thepreparation are also observed, specifically, the stimulation of thekeratinisation and the granulocytopoiesis (granulocytosis) with noinflammatory effects on the skin and the mucous membrane.

When intense transdermal absorption of the preparation is simulated,using a subcutaneous route of administration, the most characteristicsign of the systemic effect is the neurotoxicity with prevalence of CNSdepression phenomena.

Taking as a base the determination of the average toxic doses and othersin mice (TD₅₀—0.4 ml of solution at 17±3.8%, TD₁₆—0.4 ml of solution at10%, TD₈₄—0.4 ml of solution at 35% in animals), as an approximatenon-toxic dose in conditions of local cutaneous application in rats thedose of 1.0-1.5 ml/Kg of 5% solution is taken.

The extract solutions at a concentration of 5-50° have neither localirritant nor sensitizing (allergenic) properties when are cutaneouslyapplied.

Studies of Anti-Viral Activity

The objective of the present study was to examine the isolated extractof walnuts according to the first aspect used in the manufacture of apreparation (hereinafter, the extract) in order to prove the presence ofanti-viral activity (anti-herpes) in cell cultures. As an experimentalmodel the following cells were used: continue cell line DR of bonesarcoma (obtained from the virology laboratory of the National Centre ofGeorgia of especially dangerous infections) and primary culture of humanfibroblasts of musculocutaneous origin (abortive material, from theInstitute of obstetrics, gynaecology and perinatal medicine of theMinistry of Health of Georgia). Viral material—herpes simplex virus typeI, obtained from the museum of the virology laboratory of the Instituteof viral preparations of the Ministry of Health of the RussianFederation (Moscow).

In order to carry out the experiment the method of cross valuation wasused, which includes the simultaneous analysis of differentconcentrations of the extract (from 1:5 to 1:80) and of differentdilutions of the virus (from 1:10 to 1:10,000,000; traditionally, it isdesignated in the following way: from −1.0 lg to −7.0 lg). This methodpermits to show the more little nuances of the interaction between saidpreparations and the doses, as well as to show an optimal variant ofmaximum protection of the cells against the viral infection (ZdrodovskiP. F., Sokolov M. I.—Rukovodstvo po laboratornoy diagnostike virusnikh irikketsioznikh bolezney [Manual of analytical diagnostic of viral andrickettsian diseases//M., “Meditsina”, 1965, c396).

The analysis of the obtained results permitted to univocally concludethat the vegetal extract used for the manufacture of the preparation wasable to produce the inhibition of the reproduction of the herpes simplexvirus in DR cell cultures (bone sarcoma) and musculocutaneous humanfibroblasts. This effect was more intense when the extract was used in adilution of 1:5 (the pure extract was not assayed intentionally takinginto account the possibility of a citotoxic effect on the cells). Themethod of cross valuation of the extract and the virus in cell culturespermitted to demonstrate the protective anti-herpes effect of theextract and to find out some regularities of the course of the infectionin the cells.

Studies of Anti-Herpes Activity

In in-vitro studies, the isolated extract of walnuts according to thefirst aspect (hereinafter, the extract) demonstrated to have protectiveproperties against to herpes simplex virus (HSV) in cell cultures ofhuman fibroblasts and DR bone sarcoma, so that it was considerednecessary to study the anti-viral effect of the extract in white mice.

To do this, an herpes infection model in white mice was used to studythe mortality of the animals and the reproduction index of the virus.The state of the immune system of the organism was evaluated in terms ofantibody production, phagocytosis and interferon activity. Theembriotoxic and mutagenic effect of the herpes virus and the state ofthe antioxidant protection in pregnant females of mouse were alsoanalysed. All these parameters were studied in infected mice treatedwith the extract.

In conclusion, the studies demonstrated convincingly that the extractcould be used successfully to neutralize selectively theimmunodepressant effect of the herpes virus. We can argue the differentmechanisms of the properties of the extract we have indicated, but themost acceptable are the following: in presence of herpes virus, anhormonal unbalance is generated in the organism, a general and cellhypoxia is developed and the destructive processes lead to anintoxication. All these phenomena are either originated from an existingimmunopathology or caused from themselves. In other words, in thepresence of herpes virus, at least all said four factors are produced:hormonal unbalance, hypoxia, intoxication and immunopathology, withmutually aggravating effects.

One of the mechanisms of the protection effect of the extract is itsantioxidant properties, which favour the re-establishment of the breathat intracellular level (including the immunocytes). It is believed thatthe extract produces these effects by means of the active physiologicalsubstances contained in it (antibiotic, Juglone and flavonoids;micro-elements; complex of vitamins C, E, PP), which favour theintensification of the functional activity of the immunocompetent cells.

Therefore, the extract can be classified without any doubt among theactive natural remedies that can be used efficiently in the preventionand the treatment of viral and bacterial infections, the pyogenic andinflammatory diseases, as well as other pathological states whichrequire an enhancement of the metabolic and adaptive processes.

1. An isolated, stable juglone extract of walnuts, the juglone extractobtained by a process comprising the steps of: i) obtaining unripewalnut fruits as raw material; ii) preparing the raw material of step i)for extraction; iii) freezing the prepared material of step ii); iv)drying the frozen material of step iii); v) extracting the driedmaterial of step iv) by adding an alcohol to the dried material at aratio from about 1:2 to about 1:5 (weight/volume); and vi) filtering theextracted material of step v); wherein step v) is performed in a time ofabout 10 minutes or less; and wherein the extract is stable for a periodup to 24 months.
 2. The juglone extract of claim 1, wherein the extractexhibits anti-viral, anti-bacterial, and anti-fungal properties.
 3. Thejuglone extract of claim 1, wherein the extract has anti-oxidantproperties.
 4. The juglone extract of claim 1, wherein the preparationof the raw material for step (ii) comprises A) cleaning the raw materialvia mechanical means, B) washing the raw material, and C) drying the rawmaterial.
 5. The juglone extract of claim 1, wherein step (iii)comprises freezing the prepared material of step ii) at a temperature ofabout −18° C. to about −20° C. for a time period of about 70 hours toabout 170 hours.
 6. The juglone extract of claim 1, wherein step (iv)comprises A) peeling the shells of the frozen material of step iii) inthin layers of about 2 mm to about 3 mm, and B) drying the frozenmaterial of step iii) to a humidity lower than about 10%.
 7. The jugloneextract of claim 1, wherein the alcohol is ethylic alcohol (96%).
 8. Thejuglone extract of claim 1, wherein the alcohol is added at a ratio ofabout 1:3 (weight/volume).
 9. The juglone extract of claim 1, whereinstep v) is performed in a time of about 5 minutes or less.
 10. Thejuglone extract of claim 1, wherein step v) is performed in a time ofabout 2 minutes or less.
 11. The juglone extract of claim 1, whereinstep vi) is first performed in a primary depuration filter andsubsequently performed in a thin pore bactericide filter.
 12. A methodof treating a disease in a patient, said method comprising the step ofadministering to the patient a therapeutically effective dose of theextract of claim 1, wherein the disease is selected from the groupconsisting of a viral disease, a bacterial disease, and a fungaldisease.
 13. A method of treating a viral infection in a patient, saidmethod comprising the step of administering to the patient atherapeutically effective dose of the extract of claim
 1. 14. The methodof claim 13, wherein the viral infection is caused by herpes simplexvirus (HSV).
 15. The method of claim 13, wherein the viral infection isa skin infection.
 16. The method of claim 13, wherein the extractneutralizes the immunodepressant effect of the viral infection.
 17. Themethod of claim 13, wherein the extract is administered as anapplication selected from the group consisting of a topical application,an intraperitoneal application, or an oral application.
 18. The methodof claim 13, wherein the extract is administered as a solution.
 19. Themethod of claim 18, wherein the solution contains the extract at aconcentration between about 5% and about 50%.
 20. The method of claim13, wherein the extract is administered topically as a solution.